Method for the production of plant seed with modified fiber content and modified seed coat

ABSTRACT

The present invention relates to a nucleotide sequences commonly designated CJAS1 comprising a novel gene from plants. The novel gene encodes a protein that is involved in seed formation and is associated with plant defense. The invention further relates to the use of the nucleotide sequence in the sense or antisense orientation to inhibit the expression of the plant gene corresponding to the CJAS1 sequence as a means to alter seed metabolism in plants, particularly cruciferous plants, more particularly  Brassica  species, to generate seeds with reduced fiber content and/or altered seed coats. The invention also relates to similar genes expressed in other plant species.

1. FIELD OF THE INVENTION

The present invention relates to plant genes involved in the formationof the seed coat in plants and in the fibre content of seeds. Inparticular, the present invention relates to plant genes involved in theformation of seed coat and the fibre content of seeds from Brassica andother species.

2. BACKGROUND OF THE INVENTION

Plant seeds contain a number of different tissues including the embryoand cotyledons that are usually encased in a layer of thickened andlignified tissue referred to as a seed coat. In general, seed coatscontain a significant portion of the total undigestable fibre content ofplant seeds. Plant seeds with reduced fibre content provide manyadvantages for use as feed products. Thus, alteration of the seed coatcomposition, as well as alteration of the composition of the othertissues of the seed for reduced fibre content can provide an improvementfor plant seeds currently used for feed.

The seed coat provides a mechanical barrier that protects the seed priorto germination and allows the seed to remain dormant or withstandmechanical challenges. Some plant species have extremely strong seedcoats that can withstand significant mechanical and environmentalinsult. Other plant species have thinner seed coats that offer a limiteddegree of protection from mechanical damage. The nature of the seed coatis determined genetically and is typically correlated with the biologyand ecology of the plant species.

In many crop species of commercial interest, a thick seed coat isgenerally undesirable since the seed coat tends to contain a high levelof undigestible fibre and is often a waste product upon processing ofthe seed for oil, meal or other products. The seed coat contributes asignificant portion of the fibre content to plant seed meals. Thus,reduction of the seed coat is an important goal for crop improvement inmany crop species. However the importance of the seed coat for theprotection of the seed itself dictates that any reduction in seed coatstill allow for the protection of the seed from injury or damage duringseed harvesting, processing, or planting of seed. Accordingly a balancebetween seed coat size and composition and the mechanical barrierfunction the seed coat provides must be achieved.

The composition of the seed coat (or hull) is also a consideration forimprovement in many crop species, particularly those that are used forfeed. Fibre content of meals derived from plant seed is an importantconsideration for formulation of rations. Fibre levels of feed productsmust be carefully maintained for many applications since high levels ofdietary fibre are associated with poor utilization of the meal and insome cases limits the utility of the meal. The plant cell wallconstitutes the majority of dietary fibre and this fibre is composed ofa relatively limited number of starting compounds arranged in a largenumber of different final products. The matrix of the cell wallscontains most of the fibre component and this fibre is composed ofvarious polymers in both covalent and non-covalent bonds.

Examples of covalent “fibre” bonds include esterified cross-linked sugarresidues such as those found in pectins and non-cellulosepolysaccharides and cross linked lignin and extensin molecules.Non-covalent “fibre” bonds include associations in cellulose fibres andCa⁺⁺ ion bridges between pectins. The cell walls and associated “fibre”component of Brassica seeds include primary cells walls and somespecialized types. Seeds consist of three main parts, cotyledons, embryoaxis and reserve tissues. Cotyledons are generally thin-walledparenchema cells in contrast to the pericarp and testa which containthickened, lignified and suberized cell walls embedded with variousundigestable compounds. Accordingly the dehulling approach has anobvious advantage in the physical separation of the portions of the seedhigh in fibre. Although the hull is a relatively small portion of theseed on a weight basis, it does contain a significant amount of thefibre content of seeds, but further reductions in fibre content would bedesirable.

Unfortunately, the precise biochemical composition of the fibrecomponent of each of the cell types in plants has not been carried out.Therefore all “fibre” content figures tend to be generalizations and maynot accurately reflect the actual composition of the fibre component. Atthe most simple and general level, plant cell walls or fibre is composedprimarily of four complex compounds. These are cellulose, non-cellulosepolysacharides, proteins and phenolic compounds or lignins. Cellulose isa simple compound comprised of repeating glucose residues, however theactual supramolecular structure of the molecule is complex.Non-cellulose polysaccharides are comprised of acid pecticpolysaccharides, hemicelluloses and various polymers of structurallydistinct sugars. There are a number of protein components to the fibrethe largest portion of which is extensin, a unique protein that forms abackbone for the further cross linking of many compounds. Lignin ofcourse represents the primary phenolic component. For the most part,none of these components are effectively utilized by monogastric animalsin diets.

Fibre content in seeds and seed meal is generally expressed as crudefibre, acid detergent fibre or neutral detergent fibre. Crude fibre (CF)typically includes lignin, cellulosic, hemicellulosic and pectinfractions of the seed. Acid detergent fibre (ADF) typically includescellulosic and acid stable lignin fractions, while neutral detergentfibre (NDF) tends to represent lignin, cellulose and hemicellulosecomponents. Thus the term fibre can mean many different chemicalcomponents. The methods for determination of the levels of these variousfibre components are standardized according to AOAC (Association ofOfficial Agricultural Chemists) methods.

The digestability of seed meal is dependent on the composition of thefibre component. Some seed coats or hulls are highly lignified andgenerally resistant to degradation following ingestion while other seedcoats may have a composition that allows easier degradation in the gutof an animal. Thus a seed coat that is low in fibre is an importantobjective for crop improvement. Similarly, the composition of a seedcoat will influence the processing of seed for seed products such asprotein, starch or oil, seed coats that are more amenable to processingare preferred. This may include seed coats with reduced level ofpigments or seed coats with altered secondary metabolite composition.

Generally Brassica oilseed crops are processed for oil and meal by theuse of crushing techniques. Oil is extracted from seed following thedisruption of the seed and the resultant solid material is referred toas meal. Typically seed coats are found in the meal fraction, althoughit is possible to remove the seed coat (dehull the seed) beforeprocessing, removal of the hull is an additional cost and leads toadditional waste products.

In Brassica, there are numerous types of oilseed varieties, includinghigh erucic acid, rapeseed and canola quality varieties. Canola qualityvarieties are the most predominant types of oilseed Brassica speciesgrown for edible oil. Canola quality refers to a specific oilcomposition with reduced glucosinolate and erucic acid content providinga highly valuable edible oil. Brassica varieties that produce highlevels of erucic acid are grown for industrial purposes, and rapeseedlines are generally not used for edible oil production, except undercertain instances or locations where the lower quality of rapeseed oilis tolerated by market conditions. Although rapeseed is widely grown,rapeseed oil does not command the premium seen for canola quality oil.Thus, canola quality varieties are of primary value to the industry.

Many Brassica species produce seed that typically has a dark seed coat,with a few species producing seed with a yellow seed coat. The normalblack seed coat of canola quality oilseed Brassica napus impartsundesirable visual characteristics to both the oil and the meal ofcanola varieties upon typical processing of canola seed. Upon crushingof the canola seed, oil and meal fractions are isolated that arecontaminated with seed coat or pigments found within the seed coat. Theoil is dark during the initial stages of processing which makes itappear spoiled. In meal, the bits of black seed coat mixed with thelight meal make it appear to be infested with insects. Thus a seed coatthat is lighter in color can have advantages for the canola crushingindustry.

Breeding canola quality Brassica seed with reduced seed coat has been animportant objective for canola breeders. Some Brassica species have aseed coat that is yellow in color and it has been found that the yellowcolor is associated with a seed coat that is typically thinner andreduced in size. The yellow seed coat also appears to have reduced fibreand is likely more digestible due to the absence of certain pigments orsecondary metabolites commonly associated with a dark, thicker, morelignified seed coat. Meal produced from yellow seeded species ofBrassica will typically have less fibre and provide a product that has,on a percentage basis, higher protein content thus being more valuable.Accordingly, reduction of the size of the seed coat will carry manyadvantages. Thus the development of Brassica napus species with a yellowseed coat is an important goal for the Brassica oilseed industry.

Studies have been carried out with the objectives to find new sources ofgenes encoding the formation of a yellow seed coat in Brassica, forexample: Wu-JiangSheng et al., 1997, Study on a new germplasm resourceof a dominant gene controlling yellow seed coat in Brassica napus L.,Journal-of-Huazhong-Agricultural-University., 16: 1, 26-28.,Wu-JiangSheng et al., 1998, A study on the inheritance of ayellow-seeded mutant of rapeseed (Brassica napus L.).,Chinese-Journal-of-Oil-Crop-Sciences 20: 3, 6-9., Li-JiaNa et al., 1998,An initial study of the inheritance of seed colour in yellow-seededrapeseed (Brassica napus) lines with different genetic backgrounds.Chinese-Journal-of-Oil-Crop-Sciences 20: 4, 16-19. However, most of thetraits identified are not suitable for simple introgression into canolaquality Brassica breeding lines.

Other attempts to introduce a yellow seed coat into Brassica napus haveincluded the introgression of the yellow seeded trait from otherBrassica species that are being developed into edible oilseed qualitybreeding lines. Examples of these studies include: Barcikowska et al.,1997, Seed coat pigmentation—F2 yellow-seed forms of Brassica junceaCoss X B. carinata Braun. Rosliny-Oleiste 18: 1, 99-102., Meng-JinLinget al., 1998, The production of yellow-seeded Brassica napus (AACC)through crossing interspecific hybrids of B. campestris (AA) and B.carinata (BBCC) with B. napus. Euphytica. 103: 3, 329-333., Qi-CunKou etal., 1996, Studies on the transfer of yellow-seeded trait from Brassicacarinata to B. napus. Jiangsu-Journal-of-Agricultural-Sciences 12: 2,23-28. Although it is possible to obtain yellow seeded lines from theseinterspecific crosses, the resultant lines are often unstable withregards to the trait and stabilization and management of the traitduring the breeding process often proves unreliable.

Accordingly, some experiments have been carried out to to provide ameans to stabilize the trait e.g., Vyvadilova et al., 1999, The use ofdoubled haploids to stabilize yellow-seededness in oilseed rape(Brassica napus)., Czech-Journal-of-Genetics-and-Plant-Breeding. 35: 1,7-9., but the ability to routinely stabilize and obtain yellow seededvarieties is not predictable or conveniently accomplished.

Still other work has been conducted to identify molecular markers thatco-segregate with the yellow seeded trait as a means to more efficientlymanage the production of yellow seeded Brassica varieties. Theseinclude: Chen-B Y et al., 1997, Identification and chromosomalassignment of RAPD markers linked with a gene for seed colour in aBrassica campestris-alboglabra addition line., Hereditas-Landskrona 126:2, 133-138., and Deynze-A E-van, et al., 1995,The identification ofrestriction fragment length polymorphisms linked to seed colour genes inBrassica napus., Genome 38: 3, 534-542.

Still other studies have attempted to select mutation in Brassica toimpart the yellow seed color. WO9849889A1 teaches a method to selectyellow seeded characteristics from rapeseed lines through the use ofmicrospore culture and selection of mutated lines, but the resultantplants must be used for breeding into canola quality lines and this is adifficult and laborious process to carry out if the intent is to derivecanola quality lines containing a yellow seed coat.

Despite all of these studies, a convenient source of a lower fibre,yellow seeded trait in Brassica or a convenient means to manipulate thenaturally occurring trait in other Brassica species has yet to beidentified. In addition, the development of yellow seeded varieties withlow fibre content is a most preferred objective of many Brassica oilseedbreeding programs. However, this has been difficult to accomplish todate. Thus, nearly all of the Brassica napus oilseed crops commerciallygrown still have the dark-seeded characteristic and only a few havevarying degrees of yellow seeded characteristics. The fibre content ofconventional canola varieties has remained more or less constant despitethese efforts towards the production of low fibre, yellow seededvarieties. Thus, it remains an important objective for the Brassicaoilseed industry to develop yellow seeded canola varieties.

Development of a means to reduce fibre and manipulate seed coat color inrelated Brassica species, including members of the cruciferous familycan open the possibility of developing canola quality crop species fromthose species where seed coat color and characteristics areundesireable. Therefore the ability to develop crops, and in particularcruciferous crops, with reduced fibre and altered seed coats is animportant element in the further development of new oilseed and mealcrops.

3. SUMMARY OF THE INVENTION

It is an object of the present invention to provide a polynucleotidesequence encoding a protein involved in the formation of fibre in plantseeds.

It is a further object of the present invention to provide apolynucleotide sequence encoding a protein involved in the formation ofthe seed coat of plant seeds.

It is a further object of the present invention to provide a method ofmodifying a plant to reduce the fibre content of the seeds of the plant.

It is a further object of the present invention to provide a method ofmodifying a plant to alter the colour of the seeds of the plant.

It is a further object of the present invention to generate a transgenicplant with a reduced fibre content compared to the unmodified plant.

It is a further object of the present invention to generate a transgenicplant with an altered seed colour compared to the unmodified plant.

In one aspect, the present invention provides nucleic acid sequencesencoding proteins involved in the formation of typical high fibre, darkcolored seed coats in Brassica and cruciferous species. The nucleicacids of the present invention, when expressed in an antisenseorientation relative to the normal presentation can cause the reductionof fibre content, and the formation of yellow colored seed coats inBrassica varieties that normally have dark seed coats. Moreover, thenucleic acid sequences of the present invention may be expressed inother plant varieties to achieve similar results.

The present invention also provides for polynucleotide sequencesinvolved in the control of fibre content in plant seed. When thepolynucleotide sequences of the present invention are expressed inplants in an antisense orientation relative to the normal presentation,seeds are generated with reduced crude fibre content relative to seedsfrom varieties with dark seed coats.

In one aspect, the present invention provides an isolated nucleotidesequence characterized in that the isolated nucleotide is selected from:

-   -   a) SEQ ID NO: 1, or a complement thereof,    -   b) SEQ ID NO: 3, or a complement thereof,    -   c) a nucleotide sequence encoding a peptide with at least 50%,        preferably 70%, more preferably 90%, most preferably 95%        homology to a peptide encoded by the nucleotide sequence of a)        or b);

wherein said isolated nucleotide sequence or complement thereof encodesa protein or a part thereof, that alters seed development in a plantexpressing said nucleotide sequence.

In another aspect, the present invention provides an isolated nucleotidesequence characterized in that the isolated nucleotide sequence isselected from:

-   -   a) SEQ ID NO: 1, or a complement thereof;    -   b) SEQ ID NO: 3, or a complement thereof; and    -   c) a nucleotide sequence that hybridizes under stringent        conditions (65° C., 6×SSC) to a) or b);

wherein said isolated nucleotide sequence or complement thereof encodesa protein or part thereof, that alters seed development in a plantexpressing said nucleotide sequence.

Preferably, the nucleotide sequences of the present invention arederived from a cruciferous plant or a plant of the genus Brassica.

Preferably, the nucleotide sequence of the present invention ischaracterized in that expression of the nucleotide sequence in a plantreduces the fibre content of the seeds, and/or lightens the colour ofthe seeds of the plant, when compared to the seeds of an unmodifiedplant.

The present invention also encompasses isolated and purified peptidescharacterized in that the peptides are encoded by the nucleotidesequences of the invention.

In other aspects, the nucleotide sequences of the present invention maybe utilized for the generation of DNA expression cassettes, orconstructs comprising a nucleotide sequence operably linked to apromoter.

In further aspects, the present invention encompasses plant cellscharacterized in that the plant cells are transformed with theaforementioned constructs, as well as transgenic plants derived fromregeneration of the transformed plants cells into whole plants. Inpreferred embodiments, the transgenic plants of the present inventionare cruciferous plants, or plants of the genus Brassica.

In alternative embodiments, the present invention also provides for amethod for modifying the seed of a plant characterized in that themethod comprises the steps of:

-   -   (a) introducing into a plant cell capable of being transformed        and regenerated into a whole plant a construct comprising, in        addition to the DNA sequences required for transformation and        selection in plants, a nucleotide sequence in accordance with        the present invention, operably linked to a promoter; and    -   (b) recovery of a plant which contains the nucleotide sequence.        Most preferably, the method generates plants bearing seeds with        reduced fibre content and/or a lightened colour when compared to        seeds from a normal plant. In other aspects, the method may        involve the use of constructs comprising sense or antisense        orientation of the nucleotide sequences of the present        invention, relative to the promoter.

The present invention also encompasses seeds characterized in that theseeds are obtained from the transgenic plants and corresponding methodsdisclosed herein.

In particular aspects, the present invention discloses the homologousnucleotide sequences designated SEQ ID NO:1, 3, and 5 in the sequencelisting. For the purposes of the present disclosure, these sequences arecollectively termed CJAS1.

The present invention provides for the transformation of plants usingthe CJAS1 sequences disclosed, and homologues thereof. The seed of aplant can be modified by the transformation of plant cells with a planttransformation vector comprising a sense or antisense portion of theCJAS1 sequence or a double stranded RNA comprising both sense andantisense portions of the gene.

The CJAS1 sequence may also be used for identification of relatedhomologous sequences deposited in public databases through comparativetechniques well-known in the art, or for the generation of ahybridization probe for the identification of related cDNA or genomicsequences from various plant species.

The present invention further provides a method of identifying andisolating a DNA sequence substantially homologous to the nucleotidesequences disclosed in the present application, characterized in thatthe method comprises the steps of:

-   -   synthesizing a degenerate oligonucleotide primer than can        hybridize to a nucleotide sequence disclosed herein under        stringent conditions;    -   labelling said degenerate oligonucleotide primer; and

using said labelled degenerate oligonucleotide primer as a probe toscreen a DNA library for said substantially homologous DNA sequence.

The present invention also provides for the use of an isolatednucleotide sequence according encompassed by the invention, forgenerating a transgenic plant having seeds with a reduced fibre contentand/or with a lighter colour when compared to seeds of an unmodifiedplant.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Plant transformation vector comprising the CJAS1 cDNA. The arrowindicates the direction of antisense expression from the 35S promoter.

FIG. 2 a: seeds from wild-type Brassica carinata, and FIG. 2 b: seedsobtained from transgenic Brassica carinata expressing CJAS1 (SEQ IDNO: 1) in an antisense orientation.

FIG. 3: Graphic representation of the reduction of crude fibre intransgenic Brassica.

FIG. 4: Graphic representation of the reduction of acid detergent fibrein transgenic Brassica.

FIG. 5: Graphic representation of the reduction of neutral detergentfibre in transgenic Brassica.

FIG. 6 a: Sequence alignment of the 5′-ends of the original CJAS1 cDNA(SEQ ID NO: 1 from Brassica carinata), and the second homologous cDNAclone obtained from Brassica carinata (which is identical to the 5′-endof SEQ ID NO: 3, a full-length cDNA obtained from Brassica napus).

FIG. 6 b: Sequence alignment of the amino-terminal ends of the predictedpeptide sequence of the original CJAS1 gene product (SEQ ID NO: 2 fromBrassica carinata), and the predicted peptide sequence shown in SEQ IDNO: 4.

FIG. 7: Manual alignment of selected regions from SEQ ID NO: 1 with theknown peptide sequences from two domains of several class I glutamineamidotransferases (CJAS1 (SEQ ID NOS:13-15); TrpG (SEQ ID NOS:16-18);PabA (SEQ ID NOS:19-21); GuaA (SEQ ID NOS:22-24); CarA (SEQ IDNOS:25-27); PyrG (SEQ ID NOS:28-30); HisH (SEQ ID NOS:31-33); and PurL(SEQ ID NOS:34-36)).

FIG. 8: Sequence alignment of SEQ ID NO: 2 with homologous peptidesequences from Arabidopsis thaliana (SEQ ID NOS:38-42) identified from aBLAST search as described in Example 13. Also indicated is a majoritysequence derived from the alignments (SEQ ID NO:37).

5. GLOSSARY OF TERMS

Amplification of DNA/amplified DNA: “amplified DNA” refers to theproduct of nucleic-acid amplification of a target nucleic-acid sequence.Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202, and in Innis et al. (eds.), PCR Protocols: AGuide to Methods and Applications, Academic Press, San Diego, 1990.

Construct: A construct comprises a vector and an insert operativelylinked to the vector, such that the vector and insert can be replicatedand transformed as required.

Expression: The generation of a protein product derived from a DNAsequence encoding the protein, comprising a combination of transcriptionand translation.

Expression cassette: A nucleotide sequence comprising a promoter inoperable relationship with an open reading frame, or a complementthereof.

Homologous: DNA or peptide sequences exhibiting similarity to anotherDNA or peptide sequence in terms of the chemical nature, order andposition of the individual residues relative to one another in thesequence. For the purposes of this application, unless stated otherwise,homology is characterized according to BLAST search results, wherein abest-fit sequence alignment is obtained. In this way, sequencescomprising residues that are similar or identical may be aligned, andgaps provided as necessary. Homology is, therefore, expressed as apercentage of similarity or identity, wherein similarity encompassesboth similar and identical residues. Unless stated otherwise, all BLASTsearches were carried out using default parameters: e.g., gapspermitted, E-value=1, organism selected as required, filter for lowcomplexity, standard genetic code, BLOSUM62 general purpose matrix; formore information seeworldwideweb.ncbi.nlm.nih.gov/Education/BLASTinfo/tutl.html.

Identity: Comparison of homologous DNA or peptide sequences providesidentification of residues that are identical in the same relativeposition of the sequence, following best-fit alignment. For the purposesof this application, unless stated otherwise, homology, best-fitalignment and identity are calculated according to BLAST search results(BLAST searching is available, for example, from the following website:worldwideweb.ncbi.nlm.nih.gov/BLAST/). Identity is provided as apercentage, indicating the percentage of residues that are identicalalong the sequences under comparison, excluding regions of gaps betweenthe aligned sequences. BLAST searching permits a standard alignmentconfiguration to automatically take into account regions of gaps ortruncations between sequences, thereby providing a “best-fit” alignment.

Isolated: A nucleotide or peptide is “isolated” if it has been separatedfrom other cellular components (nucleic acids, liquids, carbohydrates,and other nucleotides or peptides) that naturally accompany it. Such anucleotide or peptide can also be referred to as “pure” or “homogeneous”or “substantially” pure or homogeneous. Thus, a nucleotide or peptidewhich is chemically synthesized or recombinant is considered to beisolate. A nucleotide or peptide is isolated when at least 60-90% byweight of a sample is composed of the nucleotide or peptide, preferably95% or more, and more preferably more than 99%. Protein purity orhomogeneity is indicated, for example, by polyacrylamide gelelectrophoresis of a protein sample, followed by visualization of asingle peptide band upon staining the polyacrylamide gel;high-performance liquid chromatography; or other conventional methods.The peptides of the present invention can be purified by any of themeans known in the art. Various methods of protein purification aredescribed, e.g., in Guide to Protein Purification, in Deutscher (ed.),Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, ProteinPurification: Principles and Practice, Springer Verlag, New York, 1982.

Lighter seed colour/lighter seed coat colour: a seed having a colour orhaving a seed coat colour that is lighter in colour than an averagecoloured seed from a non-transgenic unmodified plant. The term ‘lighter’is used on the understanding that a natural variation will exist in thecolour of seeds obtained from both a transgenic plant of the presentinvention, and a corresponding wild-type unmodified plant. Therefore,the term ‘lighter’ is used in consideration of an average seed colour orseed coat colour when comparing seeds obtained from transgenic andunmodified plants.

Organ: A specific region of a plant defined in terms of structure andfunction, for example in the case of a plant: a stem, a leaf, an anther,a pollen grain, or a root.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provides at least one expression control element for a geneencoding a polypeptide, and to which RNA polymerase specifically bindsand initiates RNA synthesis (transcription) of the gene.

Reduced fibre content: relates to a seed derived from a transgenic plantof the present invention having a reduced fibre content when compared toa seed derived from a corresponding non-transgenic unmodified plant. Theexpression ‘reduced fibre content’ is used on the understanding that anatural variation will exist in the fibre content of seeds obtained fromboth a transgenic plant of the present invention, and a correspondingwild-type unmodified plant. Therefore, the expression ‘reduced fibrecontent’ is used in consideration of an average seed fibre content whencomparing seeds obtained from transgenic and unmodified plants.

Stringent conditions: The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the hybridization procedure discussed in Sambrook et al.,1989, at 9.52-9.55. See also, Sambrook et al., 1989, at 9.47-9.52,9.56-9.58; Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur andDavidson, J. Mol. Biol. 31:349-370, 1968. In general, wash conditionsshould include a wash temperature that is approximately 12-20° C. belowthe calculated T_(m) (melting temperature) of the hybrid pair understudy (Sambrook et al., 1989, pp. 9-51). Melting temperature for ahybrid pair may be calculated by the following equation:T _(m)=81.5° C.−16.6(log₁₀ [Na⁺])+0.41(% G+C)−0.63(% formamide)−(600/L)where L=the length of the hybrid in base pairs.

For example, typical conditions for hybridization under stringentconditions may be 65° C. 6×SSC.

Transformation: Modification of a cell by the introduction of exogeneousDNA sequence (e.g. a vector, construct or recombinant DNA molecule).

Transgenic: A cell or organism derived from a process of cellulartransformation, wherein the cell or organism comprises the introducedexogenous DNA molecule not originally present in a non-transgenic cellor organism.

Transgenic plant: A plant or progeny thereof derived from a transformedplant cell or protoplast, wherein the plant DNA contains an introducedexogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention describes nucleic acids commonly designated CJAS1,which encode proteins involved in seed metabolism in cruciferous plants.The inventors have determined that the homologous proteins encoded byCJAS1 are involved in the defense response found in plants and CJAS1represents a new gene that shows only limited homology to genespreviously described in the art. The CJAS1 sequence represents a genealso expressed during the formation of the typical dark seed coat inBrassica. The results demonstrate that the protein encoded for by CJAS1,when expressed normally in developing seeds, is involved in theformation of the normal dark seed coat found in many Brassica species,including seed coats that are high in fibre.

In this regard, the CJAS1 cDNA, when expressed in an antisenseorientation in transformed plants that normally have dark seed coatswith high crude fibre levels, unexpectedly leads to the formation ofseed with yellow colored seed coats and/or with reduced crude fibrecontent.

Thus the present invention allows for the production of low fibre yellowseeds in Brassica varieties where dark seeded high fibre seeds aretypically observed. It is fully anticipated that this discovery allowsfor the widespread development of low fibre or low fibre yellow seededcanola Brassica napus on a wide scale not previously possible usingbreeding or mutation techniques. Moreover, other plant species areconsidered amenable to corresponding modifications.

The isolation of the CJAS1 cDNA was initially accomplished from Brassicacarinata as a result of the study of genes involved in phytoalexinbiosynthesis. Phytoalexins are found in many tissues of the plant andare often associated with cellular defense and secondary metabolism.Phytoalexins are typically induced in response to pathogens such asfungi, and can also be induced by various chemical stresses. Brassicaphytoalexins are formed as a result of plant encoded enzyme activitiesthat utilize, in part, indole glucosinolates. The biosynthesis of indoleglucosinolates has been an object of study for some time asglucosinolates are generally considered to be antinutritional in nature.

Regulation of phytoalexin biosynthesis is poorly understood, andidentification of key regulatory steps has been an objective for plantscientists for some time. Phytoalexins are often found in seed coats asthey are alleged to provide some protection against seed pathogens suchas bacteria or fungi. Although individual phytoalexins are often noteffective in controlling fungi, the complex mixture of phytoalexins,lignified tissues and other secondary metabolites in seed coats canprovide a strong barrier to seed diseases. Accordingly, phytoalexinbiosynthesis is likely to be regulated during the development of seedcoats as well as at the level of pathogen invasion, thus showingregulation at many different levels.

Presently, little data exists regarding the genes involved in thephytoalexin biosynthesis. Therefore, the inventors examined theexpression of genes induced upon induction of phytoalexin biosynthesis.Spraying plants with a solution of copper chloride (an elicitor ofphytoalexin biosynthesisis) is known to induce the production ofphytoalexins as well as other plant defense responses. Plant cells weretreated with copper chloride and cDNA libraries were made thatrepresented copper-chloride induced genes. As part of characterizing themultitude of genes that showed copper chloride induction, the cDNAs weresequenced and the sequences were compared to database entries (seeExamples).

cDNAs that were specifically induced by copper chloride treatment andnot present in the gene databanks were used in experiments withtransgenic plants wherein said transgenic plants were made with a planttransformation vector that would express the antisense strand of thecDNAs in transformed plants. The application of this technique is wellknown in the art to reduce the expression of endogenous genes in thetransformed plants and altered phenotypes can be observed.

Transformed plants containing these antisense gene vectors were visuallyexamined for altered phenotypes. As a result of this analysis,surprisingly one of the transformation vectors, comprising an antisensegene utilizing the CJAS1 cDNA (SEQ ID NO: 1) caused altered seed coatcolor. The inventors observed that over 50% of the transformants for theCJAS1 cDNA antisense produced a lighter coloured (yellow) seed, whichcontrasted to the darker seeds of the unmodified plants. These yellowseeds gave rise to plants that segregate in a 3:1 ratio for productionof yellow seed. In addition to alteration of seed coat color, thetransgenic plant seeds also showed a significant reduction in crudefibre content (see examples).

Therefore, the present invention encompasses the discovery that theinhibition of the plant gene corresponding to the CJAS1 cDNA (byantisense expression) can lead to the production of reduced fibre yellowseeds in Brassica varieties where dark seed coats are usually found (seeexamples). The genetics of segregation of the trait suggest that asingle insertion event is sufficient to confer the yellow seedphenotype. Reduced fibre content was also observed (see examples).Decreasing the expression of this gene appears to have the ability toalter both seed coat color and fibre content of seed. Accordingly, anovel method for the production of low fibre and/or yellow seeded canolafrom varieties of dark seeded canola has been discovered.

The present invention encompasses the CJAS1 cDNA sequence isolated fromBrassica carinata (SEQ ID NO: 1) as well as other homologous nucleotidesequences derived from Brassica carinata and other species of plant, andthe use of such homologous nucleotide sequences for the production oftransgenic plants comprising seeds with lighter colour and reduced fibrecontent. Such homologous sequences can be obtained by any one of thefollowing techniques:

1) DNA Library Screening

The nucleotide sequences of the present invention can be used to produce(degenerate) nucleotide probes, for the purposes of screening cDNA andgenomic DNA libraries of various plant species. Related techniques arewell understood in the art, for example as provided in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.(1989). In this way, sequences homologous to those ofthe present application are readily obtainable. For this reason, it isthe intention of the present invention to encompass polynucleotidemolecules comprising DNA sequences that encode peptides with significantsequence identity to those disclosed in the present application, whereinSEQ ID NOS: 1, 3, or 5, or parts thereof, are utilized as polynucleotideprobes to search for and isolate homologous polynucleotide molecules.Moreover, polynucleotides encoding proteins with significant sequenceidentity to those of the present application are expected to give riseto similar protein products with similar biochemical characteristics, tothose described in the present invention.

By using DNA library screening and PCR amplification techniques theinventors have succeeded in obtaining a full-length CJAS1 homologouscDNA from Brassica napus (SEQ ID NO: 3), a partial CJAS1 homologous cDNAfrom Brassica carinata (which was identical to the 5′-end of SEQ ID NO:3), as well as a partial cDNA of another homologous gene from Brassicanapus (SEQ ID NO: 5-see later). More details in this regard are providedin the examples.

2) Computer-based Homology Searches

The nucleotide and amino acid sequences disclosed in the presentapplication can be used to identify homologous nucleotide and peptidesequences via computer-based searching techniques (for example, BLASTsearches are available through the website atworldwideweb.ncbi.nlm.nih.gov/BLAST/). Such techniques are very familiarto persons of skill in the art, and can be readily utilized to identifyhomologous nucleotide and peptide sequences that may be used inaccordance with the teachings of the present application.

BLAST searches have been successfully used by the inventors to identifyCJAS1 homologues in Arabidopsis thaliana, which may be cloned and usedto generate transgenic plants comprising seeds with reduced fibrecontent or lighter colour, in accordance with the present invention.More details in this regard are provided in the Examples.

The present invention therefore encompasses DNA sequences obtained bytechniques known in the art for isolating homologous DNA sequences,wherein the techniques utilize degenerate oligonucleotide probes derivedfrom a sequence selected from SEQ ID NO:1, 3, and 5, or parts thereof.Alternatively, the techniques may utilize known sequence alignmentprograms that search databases such as Genbank for homologous nucleotideand peptide sequences. The degree of amino acid sequence homology willvary for each identified sequence. It is the intention of the presentinvention to encompass polynucleotide sequences comprising at least 50%sequence homology with regard to the peptide sequences encoded by thecorresponding polynucleotides. Without wishing to be bound by theory, itis generally expected in the art that enzymes with at least 50% homologycan be expected to have enzymatic activities that are similar in scope.In this regard, the essential structural features of the enzyme arepreserved to scaffold the conformation of the catalytic site of theenzyme. Therefore, the present invention encompasses polynucleotidemolecules derived by screening genomic and cDNA libraries of speciesother than Brassica carinata and Brassica napus, using degenerate DNAprobes derived from the sequences of the present application. Suchspecies include, but are not restricted to: other cruciferous speciesand species included in the genus Brassica.

The present invention also encompasses polynucleotide sequences obtainedby screening DNA libraries using degenerate oligonucleotide probesderived from the polynucleotides of the present invention, or fromsequence alignments derived from suitable nucleotide databases (e.g.Genbank), wherein the sequences encode peptides comprising at least 70%amino acid sequence homology to peptides encoded by SEQ ID NOS: 1, 3,and 5. In this regard, homologous proteins with at least 70% predictedamino acid sequence homology are expected to encompass proteins withactivity as those defined by the present invention, wherein disruptionof expression of the homologous proteins is expected to generate plantscomprising seeds with reduced fibre content and/or a lighter colour.Such proteins may be derived from similar species of plant.

The present invention also encompasses polynucleotide sequences encodingpeptides comprising at least 90% or 95% sequence homology to thepeptides encoded by SEQ ID NOS: 1, 3, and 5. This class of relatedproteins is intended to include close gene family members with verysimilar or identical catalytic activity. In addition, peptides with 90%to 95% amino acid sequence homology may be derived from functionalhomologues of similar species of plant, or from directed mutations tothe sequences disclosed in the present application.

It will also be understood to a person of skill in the art thatsite-directed mutagenesis techniques are readily applicable to thepolynucleotide sequences of the present invention. Related techniquesare well understood in the art, for example as provided in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.(1989). In this regard, the present inventionteaches the isolation and characterization of the DNA sequences asprovided as SEQ ID NOS: 1, 3, and 5. However, the present invention isnot intended to be limited to these specific sequences. Numerousdirected mutagenesis techniques would permit the non-informed technicianto alter one or more residues in the nucleotide sequences, thus changingthe subsequently expressed polypeptide sequences. Moreover, commercial‘kits’ are available from numerous companies that permit directedmutagenesis to be carried out (available for example from Promega andBiorad). These include the use of plasmids with altered antibioticresistance, uracil incorporation and PCR techniques to generate thedesired mutation. The mutations generated may include point mutations,deletions and truncations as required. The present invention istherefore intended to encompass corresponding mutants of the CJAS1 cDNAand genomic DNA sequences disclosed in the present application.

The polynucleotide sequences of the present invention must be ligatedinto suitable vectors before transfer of the genetic material intoplants. For this purpose, standard ligation techniques that are wellknown in the art may be used. Such techniques are readily obtainablefrom any standard textbook relating to protocols in molecular biology,and suitable ligase enzymes are commercially available.

The present invention also encompasses isolated and purified peptidesencoded by the nucleotides sequences of the present invention. Thepresent invention also encompasses polyclonal and/or monoclonalantibodies that are specific for the peptides of the present inventionand are capable of distinguishing the peptides of the present inventionfrom other polypeptides under standard conditions. Such antibodies canbe generated by conventional methods. For the preparation and use ofantibodies according to the present invention, including variousimmunoassay techniques and applications, see, e.g., Goding, MonoclonalAntibodies: Principles and Practice, 2d ed., Academic Press, New York,1986; and Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. 1988. The peptides andantibodies of the present invention can be labeled by conventionaltechniques. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent agents, chemiluminescent agents,magnetic particles, etc.

The present invention also encompasses a plant cell transformed with anucleotide sequence of the present invention, and as well as plantsderived from propagation of the transformed plant cells. Numerousmethods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

The following are examples, and are not limiting:

A. Agrobacterium-mediated Transformation: One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soilbacteria which genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant. Sci. 10: 1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8: 238 (1989). Bechtold et al., C. R.Acad. Sci. Paris Life Sciences, 316:1194-9 (1993).

B. Direct Gene Transfer: Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 .mu.m. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5: 27 (1987),Sanford, J. C., Trends Biotech. 6: 299 (1988), Klein et al.,Bio/Technology 6: 559-563 (1988), Sanford, J. C., Physiol Plant 79: 206(1990), Klein et al., Biotechnology 10: 268 (1992). See also U.S. Pat.No. 5,015,580 (Christou, et al), issued May 14, 1991; U.S. Pat. No.5,322,783 (Tomes, et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen.Genet.199: 161 (1985) and Draper et al., Plant Cell Physiol.23: 451(1982). Electroporation of protoplasts and whole cells and tissues havealso been described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51-61 (1994).

Following transformation of target cell(s) or tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety.

Alternatively, a genetic trait which has been engineered into aparticular line using the foregoing transformation techniques could bemoved into another line using traditional backcrossing techniques thatare well known in the plant breeding arts. For example, a backcrossingapproach could be used to move an engineered trait from a public,non-elite variety into an elite variety, or from a variety containing aforeign gene in its genome into a variety or varieties which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context. Oncea transgenic plant has been established, it is important to determinethe phenotype of the seeds of the plant. Accordingly, in a preferredembodiment of the invention a method is provided for modifying the seedof a plant comprising the steps of:

(a) introducing into a plant cell capable of being transformed andregenerated into a whole plant a construct comprising, in addition tothe DNA sequences required for transformation and selection in plants, anucleotide sequence in accordance with the nucleotide sequencesencompassed by the present invention, operably linked to a promoter; and

(b) recovery of a plant which contains nucleotide sequence.

It is apparent to the skilled artisan that the polynucleotide CJAS1 canbe in the antisense (for inhibition by antisense RNA) or sense (forinhibition by co-suppression) orientation, relative to thetranscriptional regulatory region, or a combination of sense andantisense RNA to induce double stranded RNA interference (Chuang andMeyerowitz, PNAS 97: 4985-4990, 2000, Smith et al., Nature 407: 319-320,2000). Other methods of gene inhibition can also be contemplated withinthe scope of the present invention.

A transcriptional regulatory region is often referred to as a promoterregion and there are numerous promoters that can be used within thescope of the present invention. A preferred promoter would be a promoterthat limits the expression of the antisense gene to seed tissue ortissue within the seed that contributes or is involved in the formationof seed coat. Suitable promoters would include the Brassica napin andcruciferin promoters, the bean phaseolin promoter or the soybeanconglycinin promoter. Alternative promoter types may include, but arenot limited to: constitutive promoters, induceable promoters,organ-specific promoters, developmental-specific promoters, strongpromoters, weak promoters etc.

Importantly, the present invention encompasses transgenic plants, andthe products and seeds thereof, which are generated by the methods ofthe present invention, wherein the nucleotide sequences of the presentinvention are transformed and expressed in the plant species of choice.The transgenic plants of the present invention are not limited withregard to plant species, provided that the transformation and expressionof the nucleotide sequence of the present invention in the plant givesrise to the desired phenotype regarding seeds with reduced fibre contentand/or lighter colour. Particularly preferred species includecruciferous varieties, and varieties of the genus Brassica. Mostpreferred plant species include Brassica carinata and Brassica napus,from which the CJAS1 homologous nucleotide sequences have thus far beenidentified.

It is also possible to use more recently developed strategies for themanipulation of the expression of CJAS1 related sequences. The use ofribozymes is within the skill ordinarily found in the art as a mechanismof reducing the expression of CJAS1 related genes. Genomic DNA libraryscreening and chromosome walking are further techniques that are wellknown to those of skill in the art as described for example in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour N.Y.(1989). Such techniques can be readily used to obtain promoter sequencefor the CJAS1 genes and to isolate the genomic DNA encoding the CJAS1polypeptide. In this way the expression of CJAS1 under normal conditionsof seed development can be evaluated. The promoter region can beisolated and studied in order to further devise methods for controllinggene expression of this and related gene families.

In terms of the function of the CJAS1 encoded protein, the derived aminoacid sequence of CJAS1 had 63% identity to a GMP synthase like proteinof A. thaliana (accession AAC63665 & AAC63681). The ORF of CJAS1 encodeda polypeptide of 250 amino acids with a calculated molecular mass of28.4 kDa. The polypeptide contained two domains with several amino acidseach typifying class I glutamine amidotransferases (GMP synthase)(KILGICFGHQ and HLFCIQGHPEYN) (SEQ ID NOS:11 and 12).

The cDNA was inserted into an expression vector and transformed into theEscherichia.coli GMP synthetase mutant ght1 (Van Lookeren Campagne etal., J. Biol. Chem. 266, 16448-16452 1991) but no complementation wasobserved. Thus, it is postulated that the protein encodes anamidotransferase activity distinct from GMP synthase that utilizes anaromatic compound for a substrate. This is analogous to a similar stepseen for the phenylpropanoid pathway, another pathway that is involvedin the formation of fibre (and the seed coat) and is a pathway thatutilizes aromatic compounds. In the phenylpropanoid pathway,L-phenyalanine, an aromatic amino acid, is a substrate for the enzymephenyalanine ammonia lyase (PAL). The enzymatic activity of PAL leads tothe formation of cinnamic acid by deamination which eventually leads tothe formation of lignin monomers. In the present case, CJAS1 may be asubunit of an enzyme that encodes a lyase activity utilized withinanother secondary metabolite pathway and reduction of the enzymeactivity (as evidenced by antisense RNA inhibition of the geneexpression) can lead to alteration in the formation of phenolic-relatedcompounds. This alteration may further affect the formation of othercompounds in the seed, in particular compounds that are known to berelated to fibre formation.

The following Examples serve to illustrate the present invention butshould not be regarded as limiting the scope of the invention in anyway.

EXAMPLE 1 Cloning of Genes Induced by Copper Chloride Treatment

In this example, differential screening of induced and non-induced plantcells was used to isolate cDNAs preferentially induced by copperchloride treatment. Brassica carinata (breeding line C90-1163,Agriculture and Agri-Food Canada Research Station, Saskatoon) were grownin Terra-Lite-Redi-Earth amended with Nutricote 14-14-14 fertilizer. Theplants were grown in a Conviron Model PGV36 growth chamber at 21/19° C.day/night temperatures with a 16 h photoperiod. Light intensity was180/μEs/m², provided by Sylvania extended service 40 W incandescentbulbs and Sylvania cool white fluorescent 215 W bulbs. Plants of thespecies Brassica carinata were treated with 5 mM copper chloride byspraying leaves until runoff. Total RNA was extracted from the leaves of4 week old Brassica carinata plants 12 hours after spraying with H₂O or5 mM CuCl₂. Poly(A)⁺ RNA was isolated from the total RNA usingoligo(dT)-cellulose resin (Life Technologies) according to publishedprocedures (Wu, W. (1997) Methods in Gene Biotechnology. Boca Raton,Fla.: CRC Press). The XhoI and PacI oligo-dT primer was used to primethe synthesis of the single-stranded cDNA from the RNA of CuCl₂-treatedleaves. Second strand synthesis and cloning were carried out accordingto the manufacturer's instructions in the λZAP II cDNA synthesis kit(Stratagene).

In vivo mass extinction was performed on the λZAP II cDNA following themanufacturer's instructions (Stratagene). The phagemid DNA was isolatedfrom 192 randomly selected colonies and approximately 100 ng of each DNAsample was dot-blotted onto duplicate Hybond-N nylon membranes (AmershamPharmacia Biotech). RNA from H₂O and CuCl₂ treated leaves wasreverse-transcribed (Superscript II) into sscDNA primed by oligo-dTfollowing the supplier's instructions (Life Technologies). The resultingDNA was labelled using High Prime (Roche Molecular Biochemicals). Thehybridization was conducted overnight at 42° C. in 50% (v/v) formamide,5× SSPE (20× SSPE, pH 7.4=174 g/l NaCl, 27.6 g/l NaH₂PO₄,H₂O, 7.4 g/lNa₂EDTA) 5× Denhardt (50× Denhardt=10 g/l ficoll approx. mol. weight 400kDa, 10 g/l polyvinylpyrrolidone m.w. 360 kDa, 10 g/l bovine serumalbumen) 0.5% (w/v) SDS and 100 μg/ml of single stranded fish DNA (RocheMolecular Biochemicals). The membranes were washed in 2×SSC (1×SSC,pH7.0=: 0.15 M NaCl, 0.015 M sodium citrate), 0.5% (W/V) SDS at roomtemperature for 15 min followed by three washes in 0.2×SSC, 0.2% (w/v)SDS at 60-65° C. for 15 min each, then exposed to X-ray film (Kodak,XAR-5) for autoradiography. Rapid amplification of 5′-cDNA ends wasperformed using the 5′-RACE System (Version 2.0) kit as described in themanufacturer's instructions (Life Technologies). The PCR products werecloned into the vector pCR2.1 (Invitrogen) for sequencing.

A number of the sequences showed similarity to known genes, particularlygenes involved in the defense response. These sequences were not studiedfurther. However, a small number of the cDNAs were not homologous toknown sequences and these cDNAs were specifically identified as uniquedefense-related sequences. The sequence of one of these cDNAs, CJAS1, isshown as SEQ ID NO:1. The derived amino acid sequence of CJAS1 is shownas SEQ ID NO:2.

EXAMPLE 2 Construction of Antisense Genes Using Defense-RelatedSequences

cDNAs that were specifically induced by copper chloride and nothomologous to any genes with known functions were used to constructantisense RNA transformation vectors. Plant transformation vectorscomprising a duplicated 35S promoter for the expression antisensesequences were used. The transformation vector was RD400 (Datla, R. S.S., Hammerlindl, J. K., Panchuk, B., Pelcher, L. E., and Keller, W.,1992, Gene 211:383-384).

The plant transformation vectors used for the construction of antisenseRNA genes were constructed by inserting (ligating) the defense-relatedcDNAs in the antisense orientation relative to the double 35S promoterusing standard vector/insert ligation techniques. The orientation of thecDNAs was confirmed by restriction mapping. The restriction map of thecDNA inserted into the vector is shown in FIG. 1.

EXAMPLE 3 Transformation of Plants with Antisense Transformation Vectors

The plant transformation vectors carrying the antisense genes made withthe defense-related cDNAs were used for plant transformation. The plantspecies used for transformation was a selection of Brassica carinata,the same selection that was originally treated with Copper chloride.This selection of B. carinata has dark colored, thick seed coats. Fortransformation, Agrobacterium tumifaciens strain GV3101/pMP90 (Koncz C.& Schell, J., 1986, Mol. Gen. Genet. 204:383-396) was used. Thetransformation vectors were introduced into the Agrobacterium strainusing standard techniques. Plant explants were co-cultured for 3 daysthen transferred to selective media. Plants were selected on kanamycin.

EXAMPLE 4 Isolation of Yellow Seeded Transformed Plants

Transformed B. carinata were analyzed by visual characterization. Offive independent cDNAs analyzed by the method of antisense RNAexpression in planta, one vector composed of the CJAS1 cDNA (SEQ IDNO: 1) in the antisense orientation produced numerous transformed plantsthat had yellow colored seed coats. The control transgenic plants, aswell as the plants transformed with the four other cDNAs hadpredominantly dark colored seed coats. A sample comparison of seedsobtain from wild-type Brassica carinata and transgenic Brassica carinataexpressing antisense CJAS1 (SEQ ID NO: 1) is shown in FIG. 2 a and FIG.2 b.

EXAMPLE 5 Segregation of the Yellow Seed Trait

Transformed plants composed of the CJAS1 cDNA in the antisenseorientation under the control of the 35S promoter were selfed and theresultant progeny grown out and observed for the presence of yellowseeds. It was found that in most plant lines a 3:1 ratio of yellow todark colored seeds were observed, indicating a single locus of insertionfor the antisense gene.

EXAMPLE 6 Fibre Content of Transformed Lines

Seed from transgenic and non-transgenic lines was analyzed for crudefibre content. Percent crude fiber, acid detergent fiber and neutraldetergent fiber methods were carried out as described in the bookDietary fiber Analysis and Applications (sections or methodsdesignations), 7.061-7.065 published by the Association of OfficialAgricultural Chemists, and National Forage Testing Associationprocedures 4.1, and 5.1. Each independently derived transgenic line isassigned a designation as an event. These events are referred to as B.carinata Events in Table 1. Out of 13 transgenic lines analyzed, 5showed a greater than 30% reduction in crude fibre content, while 4 morelines showed a statistically reduced level of crude fibre. Thus nine outof thirteen transgenic lines showed a reduction in fibre content as aresult of the expression of the CJAS1 cDNA (SEQ ID NO: 1) in anantisense orientation.

The percent crude fibre (representing the lignin, cellulosic,hemicellulosic and pectin fractions) of all the transgenic events testedwere lower than the null control (Table 1 and FIG. 3). The largestreduction in crude fibre content was observed from event 90-18-1, whichwas 36.3% lower than the null control (Table 1 and FIG. 3). Four otherindependent events 90-6-1, 90-17-1, 90-25-1 and 90-34 were found to havecrude fibre levels at least 30% lower than the null control (FIG. 3).

Table 1. Summary of the fibre analysis from greenhouse grown transgenicBrassica carinata. Values are expressed as the mean of two independentdeterminations. Percent crude fibre, acid detergent fibre and neutraldetergent fibre methods were carried out as described in AOAC7.061-7.065, NFTA 4.1, NFTA 5.1, (Designations for methods outlined bythe Association of Official Agricultural Chemists and the NationalForage Testing Association, see above).

% Crude % ADF % NDF B. Carinata Event Fibre (D.B.) (D.B.) (D.B.) NullControl 6.2 8.7 11.7 Non-Transformed Control 5.1 7.8 11.8 90-18-1 3.95.2 11.9 90-20 4.6 7.9 12.6 90-4 5.1 8.5 10.3 90-19 4.8 7.1 11.1 90-7-15.5 8.0 10.6 90-15 4.7 8.2 11.1 90-11 4.7 7.1 10.5 90-6-1 4.3 6.4 10.190-17-1 4.1 5.9 11.6 90-25-1 4.1 6.4 10.1 90-29-1 4.9 7.4 11.5 90-34-14.2 5.8 10.2 90-30-1 4.6 6.9 12.0

The acid detergent fibre content of all transgenic events tested wasreduced when compared to the null control (FIG. 4). The acid detergentfibre (ADF) analysis tends to measure the cellulosic and acid stablelignin residues that are not acid soluble. Event 90-18-1 was observed tohave a 40% reduction in ADF content when compared to the null control(Table 1 and FIG. 4). The five events (90-18-1, 90-6-1, 90-17-1, 90-25-1and 90-34) with the largest reduction in crude fibre (FIG. 3) were alsofound to have the greatest reduction in ADF content when compared to thenull control (FIG. 4). The data suggests that the acid insoluble ligninand cellulosic fractions of these five events are reduced when comparedto the null control.

The neutral detergent fibre (NDF) analysis tends to measure the lignin(insoluble and acid soluble), cellulosic, and hemicellulosic fractionspresent in the meal. Events 90-6-1, 90-25-1 and 90-34-1 had reductionsin NDF of 13.5%, 13.9%, and 13.3%, respectively (FIG. 5). Events 90-6-1,90-25-1 and 90-34-1 also had some of the largest reductions in crudefibre and ADF (FIG. 3 and FIG. 4).

EXAMPLE 7 Transformation of Brassica napus Plants with AntisenseTransformation Vectors

In this example, the plant transformation vector composed of the CJAS1cDNA (SEQ ID NO: 1) in the antisense orientation was used for planttransformation of Brassica napus that has dark colored, thick seedcoats. For transformation, Agrobacterium tumifaciens strain GV3101/pMP90(Koncz C. & Schell, J., 1986, Mol. Gen. Genet. 204:383-396) was used.The transformation vector was introduced into the Agrobacterium strainusing standard techniques. Plant explants were co-cultured for 3 daysthen transferred to selective media. Plants were selected on kanamycin.Numerous transgenic plants lines were recovered and selfed and seed coatcolor analyzed. The results are shown in Table 2 below, and illustratethat the expression of CJAS1 (SEQ ID NO: 1) in an antisense orientationis also capable of generating lighter seed coat colour in plant speciesother than Brassica carinata.

Table 2: Summary of the seed coat colour analysis from greenhouse growntransgenic Brassica napus for different transformation events. The sametransformation construct was used as described in previous examples.Seeds were individually rated by visual inspection. The average seedcolour of the majority of the tranformation event was lighter than forthe wild-type unmodified Brassica napus.

Color 1-5 Color 1-5 Insertion 1 = Black, Insertion 1 = Black, Event # 5= Yellow Event # 5 = Yellow YSXX1 2 1.0–2.0 YSXX22 6 0.5–2.0 YSXX2 51.5–2.5 YSXX23* 1 0 0.5–2.5 YSXX3 2 0.5–1.5 YSXX25 3 0.5–1.5 YSXX4 50.5–3.0 YSXX26 1 0.5–1.5 YSXXS* 3,6 3 0.5–2.5 YSXX27 3 0.5–2.0 YSXXB 20.5–2.0 YSXX29 2 0.5–1.5 YSXX9 6 1.0–2.0 YSXX3O* 4,6 3 0.5–3.0 YSXX10 30.5–2.0 YSXX32* 3 4 0.5–2.5 YSXX11 2 0.5–2.5 YSXX34 1.0–2.0 YSXX12 20.5–1.5 YSXX36 0.0–1.5 YSXX13 2 2.0 YSXX37 0.0–0.5 YSXX14* 1 0 0.5–1.5YSXX38 0.0–1.5 YSXX15 2 1.0–1.5 YSXX39 0.5–2.0 YSXX17 4 0.5–1.0 YSXX4O0.5–1.0 YSXX18 5 0.5–1.5 YSXX43 0.0–1.0 YSXX20 2 0.5–2.0 YSXX45 70.5–1.5 YSXX21* 1 3 1.0–3.0 YSXX46 0.5–1.5

EXAMPLE 8 CJAS1 Expression in B. carinata

CJAS1 gene expression was studied via Northern blot of various planttissues taken from wild-type Brassica plants (data not shown).

The CJAS1 gene was found to have a low constitutive expression in manytissues—root, stem, leaf, flower bud and silique. The amount oftranscript in flower bud and silique was slightly greater than in theother tissues. The amount of transcript was further increased by Cu,MeJA, SA and ABA treatment. Interestingly, the timing of increasedtranscript accumulation for CJAS1 differs among treatments. The rapidresponse to Cu and MeJA implies a control mechanism sensitive tomembrane damage. The activation of CJAS1 transcription by the fourdifferent compounds is unusual but not without precedent.

EXAMPLE 9 Inhibitors Effects on Expression of BcCJAS1

The effects of the nitric oxide (NO) scavenger2-phenyl-4,4,5,5-tetramethylimidazolinone-3-oxide-1-oxyl (PTIO), theprotein phosphatase type 1 and 2A inhibitor okadaic acid (OKA), theserine/threonine protein kinases inhibitor staurosporine (STAU) and theprotein translation inhibitor cycloheximide (CHX) were determined oninduction/expression of CJAS1 in Brassica carinata. The data from theseinhibitor studies (not shown) suggest that expression of CJAS1 isactively suppressed or the transcript is rapidly turned over by a labileprotein or proteins that require phosphorylation. The STAU actedsynergistically with MeJA in induction of CJAS1 expression. Also in thenon-induced material PTIO, OKA, STAU and cycloheximide all increasedCJAS1 transcript accumulation. It is difficult to understand how NOscavenging would de-repress transcription but inhibition of L-type Ca²⁺channels in rabbit glomus cells by NO was recently reported. Therefore,PTIO treatment may have indirectly led to an increase in Ca²⁺ channelactivity which in turn may have stimulated CJAS1 transcription.

These results suggest that the CJAS1 gene may be involved in normalhousekeeping functions as well as stress response.

Southern blot analysis indicated that the gene is present in 2-4 copiesin the B. carinata genome.

EXAMPLE 10 Isolation of a Second CJAS1 Homologous cDNA from Brassicacarinata

The inventors of the present application used the Invitrogen Generacer5′ RACE kit (according to the manufacturer's instructions) and RNAisolated from B. carinata leaf tissue sprayed with 5 mM CuCl₂, todetermine whether additional nucleotide sequences can be isolated thatare homologous to Brassica carinata CJAS1 (SEQ ID NO: 1). Thecorresponding protocol ensures the amplification of only full-lengthmRNA by eliminating truncated transcripts prior to the amplificationprocess. The method is mRNA cap-dependent, thereby selecting for onlythose mRNAs that are full-length and contain both a 5′ cap structure anda 3′ polyA tail. A cDNA of interest was subsequently amplified usinggene specific primers based on the initial 1044 base pair CJAS1 cDNAsequence (SEQ ID NO: 1). The primers used for the PCR amplification areshown below:

(SEQ ID NO: 6) TW1 = 5′CCTTCACGATGGTTATGTCTGTAAG3′ (SEQ ID NO: 7) TW2= 5′CTCTTACTCTGGCTATGATCTGGTGACC3′

This procedure resulted in the amplification and purification of a 409base pair cDNA fragment from Brassica carinata that was homologous toSEQ ID NO:

-   1. A sequence alignment of the 5′ end of the Generacer-derived B.    carinata product indicates that this cDNA (which corresponds to the    5′-end of SEQ ID NO:-   3) and the originally identified CJAS1 cDNA (SEQ ID NO: 1) are not    identical. In this regard, the nucleotide and peptide sequence    alignments, as shown in FIGS. 6 a and 6 b respectively, illustrate    an 86% similarity over this region with 49 nucleotides (and 5 amino    acids) differing between the two sequences in the first 409 bp. The    majority of the nucleotide differences were found in the    5′-untranslated region including an insertion of ten nucleotides in    the Generacer product not found in the original CJAS1 cDNA clone.    However, four additional nucleotides were present in the original    CJAS1 cDNA not found in the Generacer product. Three of the amino    acid changes were conservative but the remaining two were    substitutions of basic amino acids for non-polar amino acids.

Therefore a slight sequence variation was present in the two copies ofthe CJAS1 gene in Brassica carinata, with concomitant amino acidvariation. The CJAS1 gene encodes a putative glutamine amidotransferasedomain. Based on similarity to database proteins, it is likely to be asubunit of a heterodimeric enzyme.

EXAMPLE 11 Isolation of Homologs of BcCJAS1 from Brassica napus

Using total RNA isolated from Brassica napus leaves sprayed with 5 mMCuCl₂, the inventors performed the Generacer 5′ RACE protocol inaccordance with Example 10, using the same primers (SEQ ID NOS: 6 and7). In this way, the inventors succeeded in isolating a partial cDNABrassica napus nucleotide sequence which was identical to the partialCJAS1 sequence obtained from Brassica carinata in Example 10. Using thesequence of this Brassica napus product, a gene specific primer wasdesigned to amplify the full-length Brassica napus cDNA. The primersused for the amplification of the full-length Brassica napus productwere oligo-dT and the primer shown below:TW8=5′ATTGCACCTCTATCTCTGTTATCTCTT3′(SEQ ID NO: 8)

In this way, PCR amplification successfully enabled the isolation of thefull-length CJAS1 homologue from Brassica napus, and the sequence of thecDNA was characterised using standard DNA sequencing techniques. Thesequence of the full-length cDNA product is shown in SEQ ID NO: 3, andthe corresponding predicted peptide sequence is shown in SEQ ID NO: 4.

In addition to the full-length Brassica napus CJAS1 cDNA (SEQ ID NO: 3),a partial cDNA sequence was also obtained for a CJAS1-like gene usingthe following primers based on Arabidopsis thaliana genes sequence:

(SEQ ID NO: 9) TW10 = 5′CAAAAGAAGTACCTATTGTTT3′ based on gbAAC63681 (SEQID NO: 10) TW12 = 5′AAAGCATCATGTGGACTA3′ based on embCAB79773

The sequence of this additional CJAS1-like partial cDNA obtained fromBrassica napus is shown in SEQ ID NO: 5.

EXAMPLE 12 Southern Blot Analysis of B. napus

Southern blot analysis was performed (in accordance with Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y.(1989)) on Brassica napus to estimate gene copynumber, examine gene variation and determine if related genes werepresent. The DNA was digested with BamHI, EcoRI, and SalI. BamHI is theonly one of the three enzymes that cuts within the cDNA sequence and,therefore, should yield two hybridizing fragments. Each digest was runon a gel and the blot was probed with a short 5′ B. napus Generacerproduct, comprising the first 409 nucleotides of SEQ ID NO: 3. Lowstringency washes were done and the blot was developed byautoradiography.

As expected, two strongly hybridizing fragments were observed in theBamHI genomic digest lane that correspond to near identical copies ofthe CJAS1 gene. Four faintly hybridizing fragments were also observed inthis digest. One strongly hybridizing fragment was present in EcoRIdigested DNA. An additional one to two faintly hybridizing fragmentswere observed in this digest. Only a single strongly hybridizingfragment was observed in the SalI digested DNA. Therefore, CJAS1 is nota member of a closely related gene family. There could be two to fourdistantly related genes in the B. napus genome.

EXAMPLE 13 Identification of Nucleotide and Peptide Sequences withHomology to the CJAS1 Gene (and Encoded Peptide) via Computer-basedSequence Database Searching

The inventors have manually generated an alignment of the derived aminoacid sequence of CJAS1 (taken from SEQ ID NO: 1) with the G-type GATdomains of several bacterial amidotransferase, as shown in FIG. 7.Although sequence similarity clearly exists, it is apparent that theCJAS1 cDNA does not encode a synthase domain (not shown in FIG. 7).

The nucleotide and derived amino acid sequence of the CJAS1 cDNA (SEQ IDNO: 1) was also used in a BLAST search of the nucleotide and proteindatabases at NCBI using default parameters. The results revealed anArabidopsis thaliana genomic sequence from BAC F17I23 (accessionAF160182) that had 86% identity to the CJAS1 cDNA. Three other relatedproteins comprised 59-70% homology to SEQ ID NO: 1. This similarity inthe cruciferous family of plants is not unexpected. It is anticipatedthat homologous activities in other plant species can be found. Thepeptide sequence alignments for the predicted peptide sequence encodedby SEQ ID NO:1 (i.e. SEQ ID NO: 2) together with the five correspondingArabidopsis thaliana peptide sequences identified by the BLASTsearching, are illustrated in FIG. 8.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO: 6 TM1 primer-   SEQ ID NO: 7 TM2 primer-   SEQ ID NO: 8 TM8 primer-   SEQ ID NO: 9 TM10 primer-   SEQ ID NO: 10 TM12 primer

1. An isolated nucleotide sequence comprising the nucleotide sequence ofSEQ ID NO: 1 encoding the polypeptide of SEQ ID NO:
 2. 2. An isolatednucleotide sequence comprising an antisense sequence of SEQ ID NO: 1operably linked to a promoter, wherein the expression of the nucleotidesequence in a plant reduces the fiber content of seeds of the plantcompared to seeds of a wild-type or non-transgenic plant of the samespecies.
 3. An isolated nucleotide sequence comprising an antisensesequence of SEQ ID NO: 1 operably linked to a promoter, wherein theexpression of the nucleotide sequence in a plant causes seeds of theplant to have a lighter color compared to seeds of a wild-type ornon-transgenic plant of the same species.
 4. A DNA expression cassettecomprises the nucleotide sequence of claim 1 operably linked to apromoter.
 5. A DNA construct comprising a vector, wherein the vectorcomprises an antisense nucleotide sequence of SEQ ID NO: 1 operablylinked to a promoter.
 6. The construct according to claim 5, wherein thepromoter is selected from the group consisting of: constitutivepromoter, an inducible promoter, and an organ specific promoter.
 7. Aplant cell transformed with the construct according to claim
 5. 8. Atransgenic plant regenerated from the plant cell of claim 7, wherein theexpression of the nucleotide sequence in the plant causes seeds of theplant to have a lighter color compared to seeds of a wild-type ornon-transgenic plant of the same species.
 9. The transgenic plantaccording to claim 8, wherein the transgenic plant is a cruciferousplant.
 10. The transgenic plant according to claim 8, wherein thetransgenic plant is of the genus Brassica.
 11. A transgenic seed withreduced fiber content obtained from a transgenic plant, the methodcomprising: (a) transforming a plant cell with the construct of claim 5;(b) regenerating a transgenic plant from the plant cell of (a); (c)obtain a seed from the transgenic plant of (b), wherein said seedcontains said DNA construct; and wherein expression of the nucleotidesequence reduces the fiber content in said seed compared to anuntransformed seed of the same species.